Regulation of NAD(P)H oxidase growth and transcription in melanoma cells

ABSTRACT

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kB (NF-kB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kB activation and suppress cell proliferation, the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22 phox , gp91 phox  and p67 phox  components of the human phagocyte NAD(P)H oxidase, and the 91 phox  homolog NOX 4  were demon-strated in melanomas by RT-PCR and sequencing, and protein product for both p22 phox  and gp91 phox  were detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22 phox  and NOX 4 . Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22 phox  and NOX 4 . Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kB and cyclic-AMP response element consensus oligonucleotides, without affecting DNA binding activity to AP-1 or OCT- 1 .

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to PCT International Application NumberPCT/US02/41016 filed on Dec. 19, 2002 and to provisional applicationU.S. Ser. No. 60/342,839, filed on Dec. 21, 2001, which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for inhibiting NAD(P)H oxidaseenzymes in producing reactive oxygen species for growth regulation ofnormal and malignant tissues.

2. Description of Related Art

Reactive oxygen species (ROS) generated by an NAD(P)H oxidase areimportant signaling molecules for proliferation of normal cells. Therole of a signaling NAD(P)H oxidase has been most extensively exploredin vascular smooth muscle cells, where both p22^(phox) and the uniquegp91^(phox) homolog NOX1 are important for function of an NAD(P)Hoxidase activity that mediates angiotensin II-induced superoxide (O₂)formation and redox-sensitive signaling pathways (K. K. Griendling etal. (2000) Circ. Res. 86:494–501; B. Lassegue et al. (2001) Circ. Res.88:888–894). Similar but structurally distinct NAD(P)H oxidases alsoperform signaling functions in normal vascular endothelial cells (A.Gorlach et al. (2000) Circ. Res. 87:26–32; S. A. Jones et al. (1996) Am.J. Physiol. 271 (Heart Circ Physiol 49): H1626–H1634) and adventitialcells (P. J. Pagano et al. (1997) Proc. Natl. Acad. Sci. USA (1997)94:14483–14488), and the gp91^(phox) homolog NOX4 has been described inrenal tubular epithelium (M. Geiszt et al. (2000) Proc. Natl. Acad. Sci.USA 97:8010–8014; A. Shiose et al. (2001) J. Biol. Chem. 276:1417–1423),fetal tissue (G. Cheng et al. (2001) Gene 269:131–140), placenta (G.Cheng et al., supra.), and proliferating vascular smooth muscle (13.Lassegue, et al. supra.).

Like normal cells, human tumor cells also produce substantial amounts ofROS spontaneously (B. Del Bello et al. (1999) FASEB J., 13:69–79; D. J.Morre et al. (1995) Proc. Natl. Acad. Sci. USA, 92:1831–1835; T. P.Szatrowski et al. (1991) Cancer Res. 51:794–798), and evidence points toa role for these ROS in signaling neoplastic proliferation. Mitogenicsignaling through both Ras (K. Irani et al. (1997) Science275:1649–1652) and Rac (T. Joneson et al. (1998) J. Biol. Chem.273:17991–17994) is mediated by O₂ ⁻, and transfection with mitogenicoxidase NOX1 transforms normal fibroblasts (Y-A. Suh et al. (1999)Nature 410:79–82) and creates cell lines that are tumorigenic in athymicmice (R. Arnold et al. (2001) Proc. Natl. Acad. Sci. USA 98:5550–5555).The NOX1 homolog has been found expressed in the CaCo human coloncarcinoma cells (G. Cheng et al., supra; H. Kikuchi et al., (2000) Gene254:237–243; Y-A. Suh et al, supra) and HepG2 hepatoma cells (H. Kikuchiet al., supra), and gp91^(phox) expression has been demonstrated insmall cell lung cancer (D. Wang et al., (1996) Proc. Natl. Acad. Sci.USA 93:13182–13187). However, a potential role for a phagocyte-likeNAD(P)H membrane oxidase in signaling proliferation of malignantmelanoma cells has not been previously demonstrated.

S. S. Brar and co-workers reported that endogenously produced ROS signalconstitutive activation of NF-κB and cellular proliferation in M1619malignant melanoma cells. Based upon inhibition of these events by theNAD(P)H:quinone oxidoreductase (NQO) inhibitor dicumarol, S. S. Brar andco-workers speculated that cytosolic NQO might provide the enzymaticsource of electrons for reduction of membrane ubiquinone to ubiquinol,with subsequent generation of superoxide (O₂ ⁻) from molecular oxygen.However, dicumarol also inhibited growth of H596 non-small-cell lungcancer cells (S. S. Brar et al. (2001) Am. J. Physiol. Cell Physiol.280:C659–C676). H596 cells express a mutant NQO protein and haveelevated mRNA for NQO but no detectable enzymatic activity (R. D. Traveret al. (1997) Brit. J. Cancer 75:69–75).

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method of inhibitingNAD(P)H oxidase enzymes.

Another object of this invention is to provide a method for agrowth-regulatory oxidase activity in human malignant melanoma cells.

It has been found that that the generation of intracellular reactiveoxygen species in melanomas is inhibited by diphenylene iodonium,coumarin and coumarin analogs and derivatives such as dicumarol. Forexample, it has been found that dicumarol inhibits O₂ generation byisolated plasma membranes lacking a cytosolic source of NQO. M1619,other malignant melanomas and normal human epidermal melanocytes expressmRNA for the p22^(phox) membrane subunit of the NAD(P)H oxidase andmelanomas express gp91^(phox). Melanomas and melanocytes also expressthe gp91^(phox) homolog NOX4, which has been recently found in renaltubular epithelial cells and renal cell carcinomas (R. L.Shattuck-Brandt et al., (1997) Cancer Res. 57:3032–3039), glioblastomas(G. Cheng et al., supra) and CaCo colon cancer cells (G. Cheng et al.,supra). Finally, membrane O₂ generation and melanoma proliferation arereduced by inhibitor strategies directed at the leukocyte NAD(P)Hoxidase. It is thought that a form of this same enzyme system serves asa growth regulatory oxidase in malignant melanoma cells.

Consideration of the specification, including the several figures andexamples to follow will enable one skilled in the art to determineadditional objects and advantages of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the generation of intracellular reactive oxygenspecies in melanomas is inhibited by diphenylene iodonium and dicumarol.

FIG. 2 is a graph showing that plasma membranes from malignant melanomacells generate reactive oxygen species.

FIG. 3 shows proliferating malignant melanoma cells express p22^(phox),gp91^(phox), NOX4 and p67^(phox). FIG. 3A shows RT-PCR performed onnearly confluent M1619 melanoma cells and normal human epidermalmelanocytes. FIG. 3B shows immunoassays of M1619 cells for p22^(phox)and gp91^(phox). FIG. 3C shows malignant melanoma cells express NOX4.

FIG. 4 shows that antisense oliogonucleotides for NAD(P)H oxidasecomponents inhibit melanoma cell proliferation. FIG. 4A shows thatantisense oligonucleotides for p22^(phox) block melanoma growth. FIG. 4Bshows that antisense oligonucleotides for NOX4 block melanoma growth.

FIG. 5 shows NF-κB activation in melanomas is inhibited by the NAD(P)Hoxidase inhibitor diphenylene iodonium. FIG. 5A shows that theflavoprotein-dependent NAD(P)H oxidase inhibitor diphenylene iodoniumdecreases NF-κB DNA binding. FIG. 5B shows densitometry results of thep65–p50-containing bands from gels in FIG. 5A. FIG. 5C showsconstitutive nuclear translocation of NF-κB is demonstrated in M1619cells by intense brown immunohistochemical staining for p65 in nuclei.FIG. 5D shows diphenylene iodonium (50 μmol/L overnight) reducesconstitutive nuclear translocation of NF-κB. FIG. 5E shows thatdiphenylene iodonium reduces immunoreactive p65 in nuclear protein. FIG.5F shows diphenylene iodonium inhibits phosphorylation of the NF-κBinhibitor IκBα.

FIG. 6 shows that inhibition of NF-κB does not reduce melanomaproliferation. FIG. 6A shows that immunoassays of control M1619 cells orcells transduced with the adenoviral-linked superrepressor form of IκBα(AdIκBαSR). FIG. 6B shows tranduction with IκBαSR does not impairmelanoma proliferation. FIG. 7 shows NAD(P)H Oxidase inhibition reducesDNA binding to the cyclic-AMP responsive element (CRE) but not to AP-1or OCT-1.

FIG. 7A shows that proliferating M1619 melanoma cells display prominentDNA binding activity (lanes 1 and 6) for the cyclic-AMP responsiveelement (CRE). FIG. 7B shows diphenylene iodonium inhibits CRE DNAbinding activity in M1619 cells in a dose-dependent manner. FIG. 7Cshows densitometry results of EMSAs shown in FIG. 7B. FIG. 7D shows DPItreatment of M1619 cells does not inhibit DNA binding to the AP-1oligonucleotide consensus sequence. FIG. 7E shows diphenylene iodoniumtreatment of M1619 cells does not inhibit DNA binding to the OCT-1oligonucleotide consensus sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesand Figures included therein.

As used herein the following abbreviations shall mean: ROS, reactiveoxygen species; DPI, diphenylene iodonium; O₂ ⁻, superoxide anion;RT-PCT, reverse transcriptase polymerase chain reaction; NF-κB, nuclearfactor-κB; IκBα, inhibitor of NF-κB; CRE, cyclic-AMP response element;AP-1, activator protein-1; NQO, NAD(P)H quinone oxidoreductase; HEPES,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; EDTA,ethylenediaminetetraacetic acid; FBS, fetal bovine serum; EMSA,electrophoretic mobility shift assay; HBSS, Hanks' balanced saltsolution; MTT, 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazoliumbromide; DPBS, Dulbecco's phosphate-buffered saline; DMSO,dimethylsulfoxide; SOD, superoxide dismutase; PMSF, phenylmethylsulfonylfluoride; GAPDH, glyceraldehydes phosphate dehydrogenase.

This invention demonstrates evidence for a growth-regulatory oxidaseactivity in human malignant melanoma cells. More specifically, it hasbeen found that that the generation of intracellular reactive oxygenspecies in melanomas is inhibited by diphenylene iodonium, coumarin andcoumarin analogs and derivatives such as dicumarol. Using RT-PCR it wasfound that in M1619 melanoma cells for mRNA expression of the NAD(P)Hoxidase components p22^(phox), gp91^(phox), the gp91^(phox) homologNOX4, p67^(phox), and possibly p47^(phox) (FIGS. 3A and 3C). Expressionof oxidase components is not necessarily a transforming event inmelanomas, since p22^(phox) and NOX4 were also expressed in normalmelanocytes. However, several oxidase components appear criticallyimportant for malignant growth, since melanoma proliferation is reducedby transfection of antisense but not sense oligonucleotides p22^(phox)(FIG. 4A) and NOX4 (FIG. 4B). Thus, the NAD(P)H oxidase is a normalcomponent of signaling machinery that may be parasitized to servemalignant proliferation.

The putative melanoma NAD(P)H oxidase shares some of the functionalproperties of the NAD(P)H oxidase of phagocytes, but with importantdifferences. Like the phagocyte oxidase, the initial enzyme productappears to be O₂ ⁻ (FIG. 2). Also, the melanoma oxidase activity appearsto be localized in membranes rather than in the cytosol (FIG. 2). Themelanoma oxidase utilizes NADPH as its preferred substrate (FIG. 2).However, in vascular smooth muscle cells, which express gp91^(phox),NOX1 and NOX4, appear to employ only NOX1 as the functional homolog (13.Lassegue et al. (2001) Circ. Res. 88:888–894).

In phagocytes, the NAD(P)H oxidase consists of two membrane proteins,gp91^(phox) and p22^(phox) that bind a flavin adenine nucleotide (FAD)and form a cytochrome with a redox midpoint potential of −245 mV and areduced minus oxidized difference spectrum of 558 (B. M. Babior (1999)Blood 93:1464–1476). At least two and possibly three cytosolic proteins(p47^(phox), p67^(phox) and p40) are also essential and several othercytosolic components participate, including the small GTPases, Rac1 orRac2. The oxidase is thought to contain all the factors necessary fortransporting electrons from the donor substrate NADPH via FAD togenerate superoxide (O₂ ⁻) from molecular O₂. A similar oxidase has beenreported to serve a signaling function in nonphagocytic cells, where itappears to share some of the components with its phagocyte cousin, butwith critical distinctions, including a delayed time course foractivation and lower level of activity. Endothelial cells appear toexpress all the phagocyte oxidase components, including gp91^(phox),p22^(phox), p47^(phox) and p67^(phox) (A. Gorlach et al., supra; S. A.Jones et al., supra). In contrast, vascular smooth muscle cells expressp22^(phox) (K. K. Griendling et al., supra; M. Ushio-Fukui et al. (1996)J. Biol. Chem. 271:23317–23321), p47^(phox) (K. K. Griendling et al.,supra), and the unique homolog NOX1 (B. Banfi et al. (2000) Science287:138–142; B. M. Babior, supra; K. K. Griendling et al., supra; Y-A.Suh et al., supra). Yet another gp91^(phox) homolog NOX4 has beendescribed in renal tubular epithelial cells (M. Geiszt et al. (2000)Proc. Natl. Acad. Sci. USA, (2000) 97:8010–8014; A. Shiose et al. (2001)J. Biol. Chem. 276:1417–1423), fetal tissue (G. Cheng et al., supra),placenta (G. Cheng et al., supra), and proliferating vascular smoothmuscle (B. Lassegue et al., supra).

It has been found that NOX4 in normal human epidermal melanocytes and inmalignant melanoma cells, where interference with its expressionsubstantially inhibits malignant proliferation (FIG. 4B). These resultsdiffer from experiments in normal cells, where NOX4 transfectionsuppresses rather than enhances proliferation (M. Geiszt et al., supra;A. Shiose et al., supra). However, the finding of NOM in renal cellcarcinomas (A. Shiose et al., supra), glioblastomas (G. Cheng et al.,supra) and CaCo colon cancer cells (G. Cheng et al., supra), and now inmalignant melanomas, raises the possibility that this unique homologmight play an important redox signaling role necessary for malignantprosperity and progression.

A potentially large number of signal transduction and gene expressionsystems might be influenced by a growth regulatory NAD(P)H oxidase (B.M. Babior, supra), among which is the redox-regulated transcriptionfactor NF-κB. Brar, et al. have previously shown that antioxidantsreduce IκBα phosphorylation and constitutive NF-κB activation inmalignant melanoma cells (Brar et al., supra).

It has been shown that the flavoprotein inhibitor diphenylene iodoniumreduces IκBα phosphorylation (FIG. 5F) and constitutive NF-κB activation(FIGS. 5A–E), suggesting that reactive oxygen species from an NAD(P)Hoxidase contribute to constitutive NF-κB activation.

Repression of NF-κB interferes with normal and transformed cellproliferation (M. Hinz et al. (1999) Mol. Cell Biol. 19:2690–2698; B.Kaltschmidt et al. (1999) Oncogene 18:3213–32257), and inhibition ofNF-κB by antisense strategies (K. A. Higgins et al. (1993) Proc. Natl.Acad. Sci. USA 90:9901–9905) or by overexpression of the NF-κB inhibitorIκBα blocks tumor growth. In malignant melanomas NF-κB is activated as aresult of enhanced constitutive IκB kinase activity (R L.Shattuck-Brandt et al., supra) and is thought to play a significant rolein autocrine generation by melanomas of the chemokinesMGSA_(α)/GRO_(α)and interleukin-8 (J. Yang et al. (2001) Cancer Res.61:4901–4909).

It was observed from the results shown in FIG. 6 that, in contrast toresults with other tumor types, growth of M1619 melanoma cells byexpression of a superrepressor form of the NF-κB inhibitor IκBα was notsuppressed. Thus, NF-κB activation in this melanoma cell line may play agreater role in conferring resistance of the tumor to apoptosis,chemotherapy and radiation through upregulating expression ofanti-apoptotic bcl-2 family proteins (A. A. Beg et al. (1996) Science274:782–784; C—Y. Wang et al. (1999) Nature Med. 5:412–417).

As shown by the significant inhibition by diphenylene iodonium of DNAbinding to CRE (FIGS. 7B and 7C), an alternative group of transcriptionfactors that could be redox-regulated by the NAD(P)H oxidase is theATF/CREB family (O. M. Andrisani (1999) Crit. Rev. Eukaryotic GeneExpression 9:19–32; S-I. Kurata (2000) J. Biol. Chem. 275:23413–233416).Molecular disruption of ATF/CREB-mediated transcription has beenpreviously shown to reduce proliferation, metastatic potential andradiation resistance of malignant melanomas (P. M. Cox et al. (1992)Nucleic Acids Res 20:4881–4887; Z. Ronai et al. (1998) Oncogene16:523–531; D. Jean et al., supra; S. Xie et al., supra).

Others have previously shown the importance of O₂ ⁻ in mitogenicsignaling (K. Irani et al, supra; T. Joneson et al, supra), andtransfection with NOX1 transforms normal fibroblasts (Y-A. Suh et al.,supra) and creates cell lines that are tumorigenic in athymic mice (R.Arnold et al., supra). The exact contribution of autocrine ROS towardthe transformed, neoplastic condition is still unclear. However, basedupon its strategic role in melanomas and presence in other malignantcell lines (G. Cheng et al., supra; H. Kikuchi et al, supra; A. Shioseet al., supra), a membrane NAD(P)H oxidase may be fundamentallyimportant for growth signaling in a broad array of tumors. If so,NAD(P)H oxidase inhibitors might present a new strategy for cancertherapy, with coumarin analogs offering promise to this end.

It has been found that dicumarol inhibits lucigenin chemiluminescence bymelanoma plasma membranes in an in vitro system lacking cytosoliccomponents (FIG. 2), suggesting inhibition of membrane NAD(P)H oxidaseactivity. Other coumarins have previously been reported to block O₂ ⁻ bythe neutrophil NADPH oxidase (F. Bertocchi et al. (1989)Naunyn-Schmiedebergs Archiv. Pharmacol 339:697–703; M. Paya et al.(1993) Arzneimittel-Forschung 43:655–658). Furthermore, prolongedtreatment with the coumarin warfarin has been recently shown to reducesubsequent risk of cancer (S. Schulman et al. (2000) N. Engl. J. Med.342:1953–1958). This beneficial effect of warfarin has been attributedto anticoagulation (S. Schulman et al., supra; C. C. Zielinski et al.(2000) N. Engl. J. Med. 342:1991–1993), but an alternative possibilityis that certain coumarins inhibit a growth regulatory NAD(P)H oxidaseimportant for malignant cell growth.

The method of this invention uses coumarin or a coumarin analog orderivative or diphenylene iodonium as a medicament comprising a doses ofdicumarol in an amount effective to disrupt the performance of theNAD(P)H oxidase and production of its reactive oxygen species signalingproducts. The medicament doses are from 1 mg. to 500 mg. per day,preferably from about 50 mg to about 200 mg per day. The result of suchtreatment is the regulation of vascular and other smooth muscle tone,treatment of ischemia-reperfusion injury syndromes such as myocardialinfarction and stroke, lowering blood pressure, treatment of asthma andregulation of growth and proliferation of cancer.

The instant medicaments can further comprise the coumarin or coumarinanalog in a physiologically acceptable carrier for administration. Anyphysiologically buffered saline, normal saline and distilled water. By“pharmaceutically acceptable” is meant a material that is notbiologically or other wise undesirable, i.e., the material may beadministered to an individual orally without causing any undesirablebiological effects or interacting in a deleterious manner with any ofthe other components of the pharmaceutical composition in which it iscontained.

The invention further provides that the medicament can be administeredin aerosol particles, by inhalation, by intratracheal injection, byintra venous injection, by peritoneal injection. When using an aerosolit has been found particularly useful to combine the medicament withvitamin K to prevent anticoagulant activity. Aerosol particles canconsist essentially of particles less than 10 microns and preferablyless than 5 microns. Such aerosols can be provided by available jetaerosol or ultrasonic nebulizer systems in common use.

Materials. Human malignant cell lines were obtained from American TypeCulture Collection (Rockville, Md.). Human epidermal melanocytes, Medium154 and Human Melanocyte Growth Supplement (HMGS) were purchased fromCascade Biologics, Inc., Portland, Oreg. RPMI medium 1640,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),antibiotic-antimycotic (10,000 U penicillin, 10,000 μg streptomycin, and25 μg amphotericinB/ml), and trypsin-ethylenediaminetetraacetic acid(EDTA) solution were purchased from the GIBCO-BRL division of LifeTechnologies (Grand Island, N.Y.). Fetal bovine serum was purchased fromHyClone Laboratories (Logan, Utah). The intracellular oxidant sensitiveprobe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was from MolecularProbes (Eugene, Oreg.). EMSA supplies were purchased from ProMega(Madison, Wis.). Supershift antibodies were obtained from Santa CruzBiotechnology, Santa Cruz, Calif. Rabbit phospho-specific antibodies forIκBα phosphorylated at serine 32 were purchased from New England Biolabs(Beverly, MA). Protease inhibitors were from Sigma Chemical Co. (St.Louis, Mo.). All other materials were purchased from Sigma Chemical Co.(St. Louis, Mo.), unless specified.

Culture of malignant cell lines and cell culture treatments. Malignantmelanoma cell lines were cultured and passaged as described Brar, et al.(See Brar et al., supra). Human epidermal melanocytes were cultured inMedium 154 supplemented with HMGS according to supplier's instructionsand passed with 0.05% trypsin and 0.53 mmol/L EDTA. Growth rates ofmelanocytes and M1619 melanoma cells were compared by measuringproliferation as described below every 24 hours for 72 hours.Intracellular generation of ROS by melanocytes or M1619 cells wasmeasured by oxidation of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to2′,7′-dichlorofluorescein (DCF) by H₂O₂. The effect of NAD(P)H oxidaseinhibitors and blockade of the flavoprotein-dependent enzymes xanthineoxidase and nitric oxide synthetase on proliferation of malignant celllines was studied in cultures stimulated with 10% FBS and grown for 48hours before measurement of proliferation as detailed below. The effectof NAD(P)H oxidase activity on transcriptional activation was studied byincubation of 70% confluent cultures with 0–50 μmol/L of theflavoprotein inhibitor diphenylene iodonium (DPI) for 24 hours prior tomeasurement of DNA binding by electrophoretic mobility shift assay(EMSA), constitutive NF-κB nuclear activation by immunohistochemicalstaining for the p65 component, or immunoassay of levels of activetranscription factor component in nuclear protein.

Measurement of proliferation in cell cultures. Proliferation of culturedcells seeded into 24-well uncoated plastic plates (Costar) at 50,000cells per well (except where indicated) was quantitated as described byBrar et al. (Brar et al., supra) using a colorimetric method based uponmetabolic reduction of the soluble yellow tetrazolium dye3-[4,5,-dimetylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) toits insoluble purple formazan by the action of mitochondrial succinyldehydrogenase. For studies with a final cell density of less than about40,000 cells per well, direct cell counts were performed on 10 randomfields/well of Wright's-modified Geimsa stained monolayers viewed at amagnification of 40× using a 0.01 cm² ocular grid.

Measurement of reactive oxygen species generation by intact cells.Intracellular production of reactive oxygen species by M1619 cells orepidermal melanocytes was measured using oxidation of2′,7′-dichlorofluorescin diacetate (DCFH-DA) to2′,7′-dichlorofluorescein (DCF) (J. A. Royal et al., supra). DCFH-DA isa nonpolar compound that readily diffuses into cells, where it ishydrolyzed to the nonfluorescent polar derivative DCFH and therebytrapped within the cells. In the presence of H₂O₂, DCFH is oxidized tothe highly fluorescent 2′,7′-dichlorofluorescein (DCF). Approximately1×10⁶ M1619 cells or human epidermal melanocytes were incubated in thedark for 10 minutes at 37° C. with 50 mol/L DCFH-DA, harvested andresuspended in plain media. Fluorescence was analyzed using a FACScan(Becton Dickinson, Sunnyvale, Calif.) flow cytometer with excitation at488 nm and emission at 530 nm.

Measurement of reactive oxygen species generation by cell membranes. Themethod of Pagano, et al. (P. J. Pagano et al. (1997) Proc. Natl. Acad.Sci. USA 94:14483–14488), with centrifugation speeds modified accordingto the work of Mohazzab et al. (H. K. M. Mohazzab et al. (1994) Am. J.Physiol. 267(Lung Cell. Mol. Physio.) 14:L815–L822), was used to preparemembranes for measurement of ROS. M1619 cells from six near confluentT-75 flasks were harvested with Cell Dissociation Solution (Sigma),washed once with ice cold DPBS and centrifuged for 5 minutes at 675 g.The pellet was resuspended in 500 μl of ice cold Tris-sucrose buffer (pH7.1 comprised of [in mmol/L] Trizma base 10, sucrose 340, PMSF 1, EDTA 1and 10 μg/ml protease inhibitor cocktail [Sigma]) and sonicated by four15-second bursts. The cell sonicate was centrifuged at 1,475 g and 4° C.for 15 minutes in an Eppendorf microfuge to remove nuclei and unbrokencells. The supernatant was then centrifuged at 29,000 g and 4° C. for 15minutes in a Beckman Optima TL ultracentrifuge. The pellet was discardedand the supernatant was further centrifuged at 100,000 g and 4° C. for75 minutes. The pellet was resuspended in 100 μl Tris-sucrose buffer andstored at −80° C. Supernatant from the last centrifugation was alsosaved as representative of lactate dehydrogenase containing-solubleelements of cytoplasm (H. K. M. Mohazzab et al., supra). Generation ofROS was measured by SOD-inhibitable lucigenin chemiluminescence, asreported by Pagano et al. (P. J. Pagano et al., supra) in 500 μl of 50mmol/L phosphate buffer (pH 7.0), containing 1 mmol/L EGTA, 150 mmol/Lsucrose, 5 μmol/L lucigenin, 15 μg cell membrane protein, 50 μgcytosolic protein and 100 μmol/L NADH or NADPH as substrate.Chemiluminescence (in arbitrary light units) was measured using a TurnerModel 20/20D luminometer (Turner Designs, Sunnyvale, Calif.) at 30second intervals for 5 minutes with and without addition of 300 U SOD todetermine dependence of light generation upon O₂ generation. The signalwas expressed as the sum of all measurements after subtraction of thebuffer blank (A. Shiose et al., supra). DPI (50 μmol/L) and phenylarsine(1 μmol/L) were added to determine dependence of light generation uponflavoprotein- and gp91^(phox) or NOX-containing NADPH oxidase enzymaticactivity, respectively.

Reverse transcriptase polymerase chain reaction (RT-PCR) detection ofNAD(P)H oxidase components. To probe for presence of p22, gp91, p47 andp67^(phox) components of the putative analog of neutrophil NAD(P)Hoxidase, and the newly described gp91^(phox) homologs NOX1 (R. Arnold etal., supra; B. Banfi et al., supra; Y-A. Suh et al., supra) and NOX4 (G.Cheng et al., supra; M. Geiszt et al., supra; B. Lassegue et al, supra;Ashiose et al, supra), semiquantitatiave RT-PCR was performed asdescribed by Brar et al. (S. S. Brar et al. (1999) J. Biol. Chem.274:200017–200026) on triplicate near confluent cultures ofproliferating cells grown in 25-mm plastic dishes. Cell monolayers werewashed twice with DPBS and lysed with 4 mol/L guanidine thiocyanate, 25mmol/L sodium citrate, and 0.5% N-lauroylsarcosine. After scraping,lysates were sheared with four passes through a pipette. RNA wasextracted by the phenol-chloroform method (P. Chomczynski et al., (1987)Anal. Biochem. 162:56–159) and quantitated spectrophotometrically at 260and 280 nm. RNA (2 μg) was reverse transcribed using 200 units of M-MLVrevere transcriptase (Promega) in a reaction mixture containing 1 mmol/LdATP, dCTP, dGTP, and dTTP; 40 unit of RNase inhibitor; 25 μmol/L randomhexamers, 5 mmol/L MgC12, 500 mmol/L KC1, and 100 mmol/L Tris-HCl (pH8.3), in a total volume of 50 μl. The resultant cDNA was PCR amplifiedfor GAPDH, p22^(phox), gp91^(phox), p47^(phox), p67^(phox), NOX1 andNOX4 using human gene-specific sense and antisense primers based onsequences published in GenBank™:

-   -   GAPDH-5′ ACCACCATGGAGAAGGCTGG [SEQ ID NO 1];    -   GAPDH-3′ CTCAGTGTAGCCCAGGATGC [SEQ ID NO 2];    -   p22^(phox)-5′ ATGGAGCGCTGGGGACAGAAGCACATG [SEQ ID NO 3];    -   p22^(phox)-3′ GATGGTGCCTCCGATCTGCGGCCG [SEQ ID NO 4];    -   gp91^(phox)-5′ TCAATAATTCTGATCCTTATTCAG [SEQ ID NO 5];    -   gp91^(phox)-3′ TGTTCACAAACTGTTATATTATGC [SEQ ID NO 6];    -   NOXI-5′ CTGGGTGGTTAACCACTGGTTT [SEQ ID NO 7];    -   NOX1-3′ GAATCCCTAAGTGCCGTAACCA [SEQ ID NO 8].    -   NOX4-5′ TAACCAAGGGCCAGAGTATCACT [SEQ ID NO 9];    -   NOX4-3′ GGCCCTCCCACCCATAGATT [SEQ ID NO 10];    -   p47^(phox)-5′ ACCCAGCCAGCACTATGGGT [SEQ ID NO 11];    -   p47^(phox)-3′ AGTAGCCTGTGACGTCGTCT [SEQ ID NO 12];    -   p67^(phox)-5′ CGAGGGAACCAGCTGATAGA; [SEQ ID NO 13]; and    -   p67^(phox)-3′ CATGTGAACACTGAGCTTCA [SEQ ID NO 14].

PCR was carried out on a Perkin-Elmer DNA thermal cycler 480. Exceptwhere indicated, amplification was carried out for 30 cycles for GADPH,32 cycles for p22^(phox), 34 cycles for NOX4 and 36 cycles for all otherprimers at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C. for 2minutes, followed by an extension step at 72° C. for 10 minutes.PCR-amplified DNA was separated on 1.2% agarose gel, stained withethidium bromide, and visualized and photographed under ultravioletlight. PCR products from defined bands were purified with QIA quick gelextraction kits (Qiagen, Chatsworth, Calif.) and sequenced automaticallyby an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City,Calif.) using the same respective primers for sequencing as for PCR.

Immunoassay for p22^(phox), gp91^(phox), IκBα, phosphorylated IΛBα andp65 component of NF-κB. To measure protein expression of p22^(phox) andgp91^(phox) in the cytosol and in the 100,000 g plasma membrane fractionof M1619 cells, immunoassays were performed as detailed earlier (S. S.Brar et al., supra) using previously described rabbit polyclonalantibodies prepared against whole human p22^(phox) (R3179) (A. J.Jesaitis et al. (1990) J. Clin. Invest. 85:821–835) and gp91^(phox)C-terminal peptide (82089)(M. T. Quinn et al. (1989) Nature 342:198–200)at dilutions of 1:1,000. To assess nuclear translocation of thecytosolic transcription factor NF-κB, the p65 NF-κB component wassimilarly immunoassayed in nuclear protein that was isolated as outlinedbelow. For immunoassay of the NF-αB inhibitor IκBα or for IκBαphosphorylated at serine 32, the same procedure was followed aspreviously reported in Brar et al. (2001), supra) with cells lysed inboiling buffer to which 50 mM dithiothreitol (DTT) had been added as areducing agent.

Transfection protocol for p22^(phox) and NOX4 sense and antisensetreatment of M1619 cells. To transfect antisense oligonucleotides forp22^(phox), M1619 cells were cultured in 6-well plates at a density of20,000 cells per well and grown in RPMI 1640 containing 10% FBS. After24 hour wells were washed once with DPBS and 800 μl of RPMI 1640 (serum-and antibiotic-free) was added to each well. Previously reported (S.Lynn et al. (2000) Circ. Res. 86:514–519) p22^(phox), sense (5′-GGTCCTCACCATGGGGCAGATC-3′) [SEQ ID NO 15] or antisense(5′-GATCTGCCCCATGGTGAGGACC-3′) [SEQ ID NO 16] oligonucleotides (2 μg)were mixed with 5 μl LIPOFECTACE Reagent (Life Technologies) and 200 μlserum- and antibiotic-free RPMI 1640 at room temperature for 15 minutes.This mixture was then added to each well and cells were incubated at 37°C. After 6 hours the transfection mixture was gently removed andreplaced with 2.5 ml of RPMI 1640 containing 10% FBS. Cell wereincubated an additional 48 hours before staining with hematoxylin andeosin for photography or quantitation of growth with the MTT assay.M1619 cells were transfected with NOX4 sense(5′-TCGAGGAGGTCCTGTGTCGG-3′) [SEQ ID NO 17] or antisense(5′-AGCTCCTCCAGGACACAGCC-3′) [SEQ ID NO 18] oligonucleotides based ongene-specific unique sequences published in GenBank™ (Accession No. NM016931). The transfection protocol was identical to that used forp22^(phox) oligonucleotides, except that 10 μl LIPOFECTIN Reagent (LifeTechnologies) was used instead and cells were photographed using aphase-contrast microscope and a green filter.

Electrophoretic mobility shift assays (EMSAs). To assess DNA binding ofNF-_(κ)B or the cyclic-AMP response element family of binding proteins,nuclear protein was isolated and EMSAs were performed as previouslyreported (Brar, et al., supra). The consensus binding oligonucleotides,5′-AGTTGAGGGGACTTTCCCAGGC-3′ [SEQ ID NO 19] and3′-TCAACTCCCCTGAAAGGGTCCG-5′ [SEQ ID NO 20], for the p50 component ofNF-κB, 5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′ [SEQ ID NO 21] and3′-TCTCTAACGGACTGCAGTCTCTCGATC-5′ [SEQ ID NO 22] for the cyclic-AMPresponse element CRE, and 5′-CGCTTGATGAGTCAGCCGGAA-3′ [SEQ ID NO 23] and3′-GCGAACTACTCAGTCGGCCTT-5 [SEQ ID NO 24] for AP-1, and5′-TGTCGAATGCAAATCACTAGAA-3′, [SEQ ID NO 25] and3′-ACAGCTTACGTTTAGTGATCTT-5′ [SEQ ID NO 26] for OCT1 were used inbinding reactions after end-labeling by phosphorylation with [γ³²P]-ATPand T4 polynucleotide kinase. Competition experiments were performedwith 10X respective unlabeled wild-type oligonucleotide sequences, andsupershift experiments were carried out by incubating the bindingreaction with 1 μg of supershift antibody.

Immunohistochemical localization of NF-κB. Constitutive activation ofNF-κB was also studied by qualitatively assessing nuclear localizationof the p65 component by immunohistochemical staining, as described (Braret al., supra).

Transduction protocols for IκBα gene transfer. To repress activation ofNF-κB, cells were transduced with adenoviral (Ad serotype 5; Ad5)vectors that were E1a/E1b-deleted and expressed a superrepressor ofNF-κB (AdI_(κ)B_(α)SR, 2×10¹¹ plaque forming units/ml) under theregulation of the cytomegalovirus (CMV) immediate-early promoter region(R. K. Batra et al. (1999) Am. J. Respir. Cell. Mol. Biol. 21:238–245)or expressed the CMV immediate-early promoter region alone (AdCMV-3,2.05×10¹¹ plaque forming units/ml, control vector). These Ad vectorswere constructed in the Vector Core Laboratory at the Gene TherapyCenter of the University of North Carolina School of Medicine and weregenerous gifts, respectively, from Dr. A. S. Baldwin of the LinebergerCancer Center and Dr. A. Ghio of the U.S. EPA Human Health EffectsCenter, Chapel Hill, N.C. Transduction was performed using previouslypublished protocols (R. K. Batra et al., supra). M1619 cells were seededonto 24-well plates at a density of 25,000 cells/well and grown for 6hours in RPMI 1640 with 10% FBS. Media was removed and replaced with 200μl complete medium containing approximately 1.25×10⁶ to 2.0×10⁷ colonyforming units of AdIκBαSR or AdCMV-3. After overnight incubation, thevector containing media was removed, and cells were washed once withwarm DPBS and reincubated with fresh complete media After an additional24 hour, proliferation was assessed with the MTT assay.

Statistical analysis. Data are expressed as mean values±standard errorfor a minimum number of four observations, unless indicated. Differencesbetween two groups were compared using the unpaired Student's t test.Two-tailed tests of significance were employed. Differences betweenmultiple groups were compared using one-way analysis of variance. Thepost-hoc test used was the Newman-Keuls multiple comparison test.Significance was assumed at p<0.05.

The present invention is more particularly described in the followingexamples which are intended as illustrative only, since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLE 1

Malignant Melanonza Cells Produce Intracellular Reactive Oxygen Species.

Intracellular production of reactive oxygen species by M1619 cells wasmeasured using oxidation of 2′, T-dichlorofluorescin diacetate (DCFH-DA)to 2′, 7′-dichlorofluorescein (DCF) (J. A. Royall et al. (1993) Archiv.Biochem. Biophys. 302:348–355). DCFH-DA is a nonpolar compound thatreadily diffuses into cells, where it is hydrolyzed to thenonfluorescent polar derivative DCFH and thereby trapped within thecells. In the presence of H₂O₂, DCFH is oxidized to the highlyfluorescent 2′,7′-dichlorofluorescein (DCF). Approximately 1×10⁶ M1619cells were incubated in the dark for 10 minutes at 37° C. with 50 μmol/LDCFH-DA, harvested and resuspended in plain media. Fluorescence wasanalyzed in approximately 10,000 cells each using a FACScan (BectonDickinson, Sunnyvale, Calif.) flow cytometer with excitation at 488 rimand emission at 530 nm.

FIG. 1 shows a comparison to spontaneous fluorescence of cells withoutDCFH treatment (blue-shaded areas) wherein M1619 melanoma cells oxidizedDCFH to DCF. Pretreatment of cells for 15 minutes with diphenyleneiodonium (DPI;50 μmol/L) or dicumarol (250 μmol/L) reduced DCFHoxidation to DCF. Treatment with DPI or dicumarol in the absence of DCFHdid not result in significant cellular fluorescence within the monitoredspectrum (data not shown). Mean fluorescence=754 untreated, 408 DPI, 396dicumarol; median fluorescence 685 untreated, 355 DPI, 359 dicumarol.

These results show that during proliferation, melanomas and melanocytesproduce intracellular reactive oxygen species, oxidant generation isblocked by DPI and dicumarol, and growth is disrupted by antioxidantstrategies.

EXAMPLE 2

Malignant Melanoma Cell Membranes Produce Reactive Oxygen Species.

In this example the source of O₂ ⁻ generation was studied by preparingplasma membrane and cytosolic fractions from proliferating M1619melanoma cells. The plasma membrane and cytosolic fractions wereexamined for their respective abilities to support SOD-inhibitablelucigenin chemiluminescence.

The 100,000 g membrane fraction of proliferating M1619 melanoma cellswas prepared as described above. Chemiluminescence stimulated by 15 ggmembrane protein was measured after incubation for 5 minutes in thepresence of 5 μmol/L lucigenin and 100 μmol/L NADH or NADPH, with andwithout addition of 50 μg cytosolic protein, 300 units/ml SOD, 50 μmol/Ldiphenylene iodonium (DPI), 1 μmol/L phenylarsine oxide or 50 μmol/Ldicumarol. M=membrane; C=cytosol; M+C=membrane+cytosol;SOD=membrane+SOD; DC=membrane+dicumarol; PArs=membrane+phenylarsineoxide; DPI=membrane+DPI; n=4 for each observation; *P<0.001 vsmembranes+NADPH.

As shown in FIG. 2 plasma membranes alone significantly increasedSOD-inhibitable lucigerin chemiluminescence, without addition of cellcytosol. When lucigenin was used at a concentration of 5 μmol/L toprevent artifactual redox cycling, the preferred substrate forgeneration of O₂ ⁻ was NADPH rather than NADH (column 1 vs 2). Cytosolfrom M1619 cells did not support chemiluminescence (column 4),mitigating against the cytosolic enzyme NQO1 as the source driving O₂ ⁻generation in this cell line. Also, addition of cytosol to membranes andNADPH did not increase light emission. Chemiluminescene wassignificantly inhibited by SOD, and by addition of the NADPH oxidaseinhibitor phenylarsine oxide and the flavoprotein inhibitor diphenyleneiodonium (DPI).

These results show that light emission is the result of O₂ ⁻ generatedby a membrane NAD(P)H oxidase. Both phenylarsine oxide (at 10–50 μmol/L)and DPI (at 10–50 μmol/L) also significantly inhibited M1619 malignantmelanoma cell growth (96.2±0.6% and 86.0±0.5 inhibition after 48 hours,respectively, at the highest dose of each; P<0.001), but melanoma growthwas unaffected (data not shown) by inhibitors of the other flavoproteinoxidases xanthine oxidase (allopurinol, 1 mmol/L) or nitric oxidesynthetase (ω-nitro-L-arginine, 100 μmol/L). Chemiluminescencegeneration was likewise reduced by direct addition of dicumarol to themembrane preparation, in the absence of cytosol. These results show thatdicumarol inhibits plasma membrane NAD(P)H oxidase activity.

EXAMPLE 3

Malignant Melanoma Cells and Melanocytes Express NAD(P)H OxidaseComponents that are Necessary for Proliferation.

To determine whether components of the NAD(P)H oxidase-neutrophil-or itshomologs are also expressed in melanomas and non-malignant melanocytes,RT-PCR on RNA extracted from proliferating cells stimulated by 10% FBS(melanomas) or HMGS (melanocytes) were performed and the results shownin FIG. 3.

In FIG. 3A the gels represent RT-PCR at 36 cycles. M1619 melanoma cells(FIG. 3A, top panel, lane 2) and other malignant melanomas (Table 1)strongly expressed the alpha subunit of cytochrome b₅₅₈, p22^(phox),initially detected at 32 cycles. The 252 base pair PCR product obtainedhas been sequenced and is identical to bases 221–372 of the reportedhuman mRNA sequence (Accession No. XM 008040).

Proliferating human melanoma cells also expressed gp91^(phox) (toppanel, lane 3, and Table 1 detectable only after 36 cycles as a 557 basepair product that was sequenced and found corresponding to bases 630 to1158 of the reported human mRNA sequence; Accession No. NM 000397).Normal melanocytes expressed p22^(phox) but not gp91^(phox).Surprisingly, M1619 melanoma cells strongly and melanocytes modestlyexpressed the gp91^(phox) homolog NOX4 (top and second panels, lane 7),detected using the primers NOX4-5′ TAACCAAGGGCCAGAGTATCACT; NOX4-3′GGCCCTCCCACCCATAGATT [SEQ ID NO 10]. The 564 base-pair productcorresponded to sequences corresponding to bases 197 to 741 of thereported human mRNA sequence (Accession No. 016931). No NOX1 homolog wasfound in either melanomas or melanocytes (lane 6, 36 cycles). Thep67^(phox) cytosolic component was also detected in melanomas (toppanel, lane 5, 36 cycles, 727 base pairs, with sequences identical frombases 556 to 1283 of the reported human mRNA sequence; Accession No. BC001606), and faint PCR product was found in melanomas for p47^(phox)(top panel, lane 4, 36 cycles). GAPDH is shown in lane 1. PCR productswere compared to that from an equal amount of mRNA from human PMNs(third panel, for p22^(phox), gp91^(phox), p67^(phox) and p47^(phox)) orCaCo colon carcinoma cells (bottom panel, for NOX1 and NOX4),respectively, as shown. RT-PCR was performed using human gene-specificsense and antisense primers based on sequences and conducted as detailedin the text and in Table 1.

By immunoassay, both p22^(phox), and gp91^(phox) were easily detectablein the 100,000 g plasma membrane fraction of M1619 melanoma cells (FIG.3B). No evidence was found for the NOX1 homolog of gp91^(phox) (FIG. 3A,top panel, lane 6). However, NOX4 was easily detectable at 34 cycles inM1619 and other malignant melanoma cells (FIG. 3A, top panel, lane 7,and 3C, lanes 1–5) as a 564 base-pair product, with sequencescorresponding to bases 197 to 741 of the reported human mRNA sequence.(Accession No. NM 016931).

Melanocytes also expressed p22^(phox) (FIG. 3A, second panel, lane 2)and NOX4 (FIG. 3A, second panel, lane 7), but did not contain mRNA forgp91^(phox) (FIG. 3A, second panel, lane 3). The p67^(phox) cytosoliccomponent was observed in M1619 cells (FIG. 3A, top panel, lane 5; 747base pairs, with sequences identical from bases 556 to 1283 of thereported human mRNA sequence; Accession No. BC 001606) and two othermalignant melanoma cell lines (Table 1). Faint PCR product was alsodetected in M1619 cells for the p47^(phox) cytosolic component of theleukocyte NADPH oxidase (FIG. 3A, top panel, lane 4), but this productwas not sufficiently well-expressed to be sequenced. Neither p67^(phox)nor p47^(phox) were found in epidermal melanocytes. Thus, three knownmembrane components (p22^(phox) and two possible partners gp91^(phox)and NOX4) and the p67^(phox) cytosolic component of the NAD(P)H oxidaseare present in proliferating M1619 and other melanoma cells, andp47^(phox) may be expressed at low levels. In contrast, whenproliferating, normal melanocytes express only p22^(phox) and NOX4.

In FIG. 3B the immunoassays of M1619 cells demonstrated that the 100,000g membrane fraction from triplicate preparations was full sizep22^(phox) and gp91^(phox). A second lighter band was seen inimmunoassays for gp91^(phox) that may represent unglycosylated protein.In FIG. 3C shows malignant melanoma cells expressing NOX4. RT-PCR wasperformed for 34 cycles using the gene-specific primers as used above.NOX4 was strongly expressed in the malignant melanoma cell lines M1619(lane 1), M1585 (lane 2), RLW1495 (lane 3), CMC 9212 (lane 4) and RLW537 (lane 5).

To begin probing the role of individual NAD(P)H oxidase components inmelanoma proliferation, sense and antisense oligonucleotides forp22^(phox) mRNA were transfected into growing M1619 cells. A wide rangeof commercially available transfection reagents all produced sometoxicity to the cells, including the transfection reagents that wereultimately employed. Nevertheless, M1619 cells treated with p22^(phox)antisense oligonucleotides had significantly slower growth subsequent totreatment than did identical cells transfected with senseoligonucleotides for p22^(phox) (FIG. 4A). Thus, p22^(phox) and NOX4appear to play roles in growth signaling for this melanoma cell line.

The results are shown in FIG. 4A wherein antisense oligonucleotides forp22^(phox) block melanoma growth. M1619 cells (20,000 cells per well)were seeded into 6-well plastic plates and grown for 24 hours.Oligonucleotides were transfected into cells using Lipofectase®, asdetailed above. After 6 hours of exposure, transfection solution wasreplaced with complete media and cells were grown an additional 48hours. Proliferation was measured by the MTT assay in triplicateexperiments. Compared with sense oligonucleotides, treatment withp22^(phox) antisense oliogonucleotides significantly reduced subsequentgrowth (* P<0.01 vs respective sense oligonucleotides). Hematoxylin- andeosin-stained M1619 cells treated with sense (top) or antisense (bottom)oligonucleotides for p22^(phox) are shown at right. As did several othertransfection reagents, Lipofectase® alone also inhibited proliferationcompared to untreated cells (55 f 4% for Lipofectase; P<0.01).

Melanoma cells transfected with NOX4 antisense oligonucleotides, withthe exception that Lipofectin® was used. These cells also hadsignificantly slower growth subsequent to treatment than did cellstransfected with sense oligonucleotides for NOX4 (FIG. 4B). Comparedwith sense oligonucleotides, treatment with NOX4 antisenseoliogonucleotides significantly reduced subsequent growth (*P<0.01 vsrespective sense oligonucleotides). Phase contrast micrographs are shownat right of p. cells treated with sense (top) or antisense (bottom)oligonucleotides for NOX4. As did Lipofectase® or Lipofectin® alone alsoinhibited proliferation compared to untreated cells (21 f 1% forLipofectin; P<0.01).

Using the same primers expression of p22^(phox), gp91^(phox) andoccasionally p67^(phox) by other malignant cell lines was detected,including small cell and non-small cell lung cancers, and ovarian,breast and prostate adenocarcinomas (Table 1). Prostate LnCap carcinomaexpressed the NOX1 homolog. H82 small cell carcinoma strongly and H520squamous cell lung cancer weakly expressed NOX4 (data not shown).

RT-PCR was performed using human gene-specific sense and antisenseprimers based on sequences published in GenBank™ as shown by [SEQ ID NO1] through [SEQ ID NO 14]. PCR was carried out for 30 cycles for GAPDH,32 cycles for p22^(phox) and 36 cycles for all other primers, withamplification at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C.for 2 minutes, followed by an extension step at 72° C. for 10 minute.PCR-amplified DNA was separated on 1.2% agarose gel, stained withethidium bromide, and visualized and photographed under ultravioletlight. The results are shown in Table 1.

TABLE 1 RT-PCR EXPRESSION OF NAD(P)H OXIDASE COMPONENTS IN HUMAN NORMALAND MALIGNANT CELL LINES GAPDH p22 gp91 p47 p67 NOX1 Growth Number ofPCR Cycles 30 32 36 36 36 36 Size of PCR Product 528 bp 252 bp 527 bp727 bp 747 bp 663 bp Human neutrophils + + + + + − Malignant MelanomasM1585 + − + − + − Slow CMC 9515 + + +/low − − − Intermediate CMC9601 + + +/low − − − Intermediate CMC 9703 + + +/low − − − IntermediateCMC 9710 + + +/low − − − Intermediate CMC 9128 + + +/low − − −Intermediate CMC 0040 + + +/high − + − Fast CMC 0056 + + +/high − − −Fast RLW 836 + + +/high − − − Intermediate RLW 1379 + + +/high − − −Fast RLW 1402 + + +/low − − − Intermediate RLW 1495 + + +/low − − −Intermediate Ovarian carcinoma + + +/high − − − Intermediate BreastMDA-MB-453 + + variable − − − Intermediate Prostate LnCap carcinoma + −− +/low − + Slow Prostate PC3 carcinoma + + +/high +/low − − SlowProstate DU145 carcinoma + + +/high +/low − − Slow Small cell lung H82cancer + + +/high +/low − − Intermediate Squamous lung H520 cancer + ++/high +/low + − Slow Adenosquamous lung H596 cancer + + +/high +/low +− Intermediate

EXAMPLE 4

NF-κB is Constitutively Expressed in Melanoma Cells and may be Regulatedby the NAD(P)H Oxidase.

The flavoprotein-dependent NAD(P)H oxidase inhibitor DPI reducedconstitutive activation of NF-κB in melanoma cells, studied by NF-κB DNAbinding activity (FIGS. 5A and B), immunohistochemically detectable p65in nuclei (FIG. 5C vs FIG. 5D) and immunoassay of the p65 NF-κBcomponent in nuclear protein (FIG. 5E).

As shown in FIG. 5A the flavoprotein-dependent NAD(P)H oxidase inhibitordiphenylene iodonium (DPI) decreases NF-κB DNA binding. Near confluent(70%) cultures of M1619 cells (n=3 per group) were incubated overnightwith or without 50 μmol/L DPI. Cells were then lysed, nuclear proteinwas isolated, and EMSAs using ³²p-labeled NF-κB consensusoligonucleotides. The arrow shows the p65/p50-containing dimer.Constitutive NF-κB DNA binding of melanoma cells was greatly reduced inDPI-treated cells (lanes 4–6), compared with cells incubated in growthmedium alone (lanes 1–3). In FIG. 5B the densitometry results of thep65–p50-containing bands from gels in FIG. 5A is shown. *P<0.001compared with no DPI.

In FIG. 5C constitutive nuclear translocation of NF-κB is demonstratedin M1619 cells by intense brown immunohistochemical staining for p65 innuclei. Confluent cells were fixed in paraformaldehyde, permeabilized,stained using an antibody to the p65 component of NF-κB and astreptavidin-biotin-immunoperoxidase based method outlined in the text,viewed under light microscopy using a blue filter to enhance contrast,and photographed at x400 magnification. FIG. 5D illustrates that DPI (50μmol/L overnight) reduces constitutive nuclear translocation of NF-κB.Compared with the intense brown nuclear staining for p65 seen in FIG.5C, DPI-treated M1619 cells demonstrate little anti-p65 brown stainingin nuclei. Nucleoli are recognizable in DPI-treated cells (FIG. 5D) butare only occasionally visible in untreated control cells (FIG. 5C).

FIG. 5E demonstrates that DPI reduces immunoreactive p65 in nuclearprotein. Near confluent (75%) cultures of M1619 cells (n=3 per group)were incubated overnight with (lanes 4–6) or without (lanes 1–3) 50μmol/L DPI. Nuclear protein was isolated and immunoassays were performedfor the p65 component of NF-κB. DPI treatment of melanoma cells alsodecreased phosphorylation of the NF-κB inhibitor Iκbα (FIG. 5F). Nearconfluent (75%) cultures of M1619 cells (n=3 per group) were incubatedovernight with (lanes 4–6) or without (lanes 1–3) 25 μmoles/L DPI. Cellswere lysed and immunoassays were performed using a phospho-specificantibody for IκBα phosphorylated at serine 32.

These findings suggest that a flavoprotein-containing NAD(P)H oxidasemay play a role in stimulating constitutive NF-κB transcriptionalactivity in these cells through generation of ROS.

EXAMPLE 5

Inhibition of NF-κB does not Impair Melanoma Proliferation.

Inhibition of NF-κB by antisense strategies reduces tumorigenicity offibrosarcomas (K. A. Higgins et al., supra.), and overexpression of theNF-κB inhibitor IκBα blocks tumor cell growth of Hodgkin's disease (R.C. Bargou et al. (1997) J. Clin. Invest. 100:2961–2969), squamous celllung cancer (R. K. Batra et al., supra), squamous cell head and neckcancer (D. C. Duffey et al. (1999) Cancer Res. 59:3468–3474) and breastcancer cells. (M. A. Sovak et al. (1997) J. Clin. Invest.100:2952–2960).

M1619 cells were infected with an adenoviral vector encoding asuperrepressor version of the NF-κB inhibitor IκBα (AdIκBαSR) todetermine if selectively inhibiting NF-κB could reduce M1619 melanomacell proliferation. Cells were grown to near confluence on 25-mm Petridishes, then transduced with 2.5×10⁶ to 1.0×10⁷ colony forming units(CFU) of AdIκBαSR. After 24 hours, cells were lysed and immunoblots wereperformed for IκBα. The IκBαSR form of IκBα is seen as a band (arrow)slightly heavier than native IκBα. The results of infection withAdIκBαSR resulted in dose-related increases IκBαSR expression are shownin FIG. 6A.

However, infection with even the highest dose (1×10⁷ colony formingunits) of AdIκBαSR did not substantially reduce M1619 melanomaproliferation (FIG. 6B), especially compared to the profound growthinhibitory effect shown by antioxidants and NAD(P)H oxidase inhibitorsin FIG. 1. M1619 melanoma cells were seeded onto 24-well plates at adensity of 25,000 cells/well and grown for 6 hours in RPMI 1640 and 10%FBS. Media was removed and replaced with 200 μl complete mediumcontaining 1×10⁷ colony forming units of the adenoviral-linkedsuperrepressor form of IκBα (AdIκBαSR) or the adenoviral vector linkedto the CMV promoter (AdCMV-3). After overnight incubation, the vectorcontaining media was removed, and cells were washed once with warm DPBSand reincubated with fresh complete media. After an additional 24 hours,proliferation was quantitated with the MTT assay. Both AdIκBαSR andAdCMV-3 slightly reduced proliferation, but neither had the profoundinhibitory effect shown by antioxidants or NAD(P)H oxidase inhibitors inFIG. 1. *P<0.05 vs FBS FIG. 7. NAD(P)H oxidase inhibition reduces DNAbinding to the cyclic-AMP responsive element (CRE) but not to AP-1 orOCT-1.

The results show that oxidant generation by a growth regulatory NAD(P)Hoxidase regulates proliferation through other signal transductionpathways.

EXAMPLE 6

Inhibition of NAD(P)H Oxidase in Melanoma Cells Reduces DNA BindingActivity for the Cyclic-AMP Response Element (CRE).

Another family of redox responsive transcription factors important formelanoma proliferation are ATF/CREB proteins that bind the cyclic-AMPresponse element (CRE). The transcription factor CREB (for CRE-bindingprotein) and its associated family member ATF-1 promote tumor growth,metastases and survival through CRE-dependent gene expression (S. Zie etal., supra), and expression of the dominant negative KCREB construct inmelanoma cells decreases their tumorigenicity and metastatic potentialin nude mice (D. Jean et al. (1998) J. Biol. Chem. 273:24884–24890).

The inhibition of NAD(P)H oxidase in melanoma cells was explored todetermine if DNA binding of transcription factors to CRE was reduced. Asreported previously for the McWo melanoma cell line (D. Jean et al.,supra; S. Xie et al., supra), proliferating M1619 cells displayedprominent DNA binding activity for CRE, comprised of ATF-1, ATF-2 andCREB-1 transcription factors. Supershift experiments with specificantibodies demonstrate that the CREB (for CRE binding protein) familymembers ATF-1 (lane 2), ATF-2 (lane 3) and CREB-1 (lane 4) contribute toDNA binding activity for CRE. A competition experiment is shown atright, in which addition of 10X molar excess unlabeled CRE (lane 7) butnot AP-1 (lane 8) eliminates DNA binding activity for CRE in melanomanuclear protein. The results in FIG. 7A show that proliferating M1619melanoma cells display prominent DNA binding activity (lanes 1 and 6)for the cyclic-AMP responsive element (CRE).

Treatment of proliferating M1619 cells overnight with DPI inhibitsCRE-binding activity in a dose-dependent manner (FIGS. 7B and 7C), butdoes not change DNA binding activity of nuclear protein for thetranscription factors AP-1 (FIG. 7D) or OCT-1 (FIG. 7E). Near confluentcultures of M1619 cells were treated overnight with DPI, and nuclearprotein was harvested for EMSAs. Compared to that in untreated cells(lanes 1–3), treatment with DPI inhibits DNA binding of nuclear proteinto CRE in a dose-dependent manner (lanes 4–6, 10 μmoles/L; lanes 7–9, 15μmoles/L; lanes 10–12, 20 μmoles/L). The results in FIG. 7B show thatDPI inhibits CRE DNA binding activity in M1619 cells in a dose-dependentmanner. In FIG. 7C the densitometry results of EMSAs of FIG. 7B areshown. *P<0.001 vs untreated cells.

In FIG. 7D the results show that DPI treatment of M1619 cells does notinhibit DNA binding to the AP-1 oligonucleotide consensus sequence.Untreated cells, lanes 1–3; cells treated with 10 μmoles/L, lanes 4–6;cells treated with 15 moles/L, lanes 7–9; cells treated with 20μmoles/L, lanes 10–12. In FIG. 7E the results show that DPI treatment ofM1619 cells does not inhibit DNA binding to the OCT-1 oligonucleotideconsensus sequence. Untreated cells, lanes 1–3; cells treated with 10μmoles/L, lanes 4–6; cells treated with 15 μmoles/L, lanes 7–9; cellstreated with 20 μmoles/L, lanes 10–12.

The results in FIG. 7 show that NAD(P)H oxidase inhibition reduces DNAbinding to the cyclic-AMP responsive element (CRE) but not to AP-1 orOCT-1. Throughout this application, various publication are referenced.The disclosures of these publications are hereby incorporated byreference into this application in order to more fully describe thestates of the art to which this invention pertains.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

1. A method for inhibiting NAD(P)H oxidase enzymes comprising treating apatient in need thereof with from 1 mg to 500 mg per day an amount of adicoumarol effective to disrupt performance of the oxidase andproduction of its reactive oxygen species signaling products, whereinsaid treatment includes the treatment of ischemia-reperfusion injurysyndromes such as myocardial infarction and stroke, lowering bloodpressure, treatment of asthma and regulation of growth and proliferationof malignant melanoma cells.
 2. The method for inhibiting NAD(P)Hoxidase enzymes according to claim 1 wherein the amount of a dicoumarolis from 50 mg to 200 mg per day.
 3. The method for inhibiting NAD(P)Hoxidase enzymes according to claim 1 wherein dicoumarol is administeredin an aerosol.
 4. The method for inhibiting NAD(P)H oxidase enzymesaccording to claim 1 wherein dicoumarol is administered orally.
 5. Amethod for inhibiting NAD(P)H oxidase enzymes comprising treating apatient in need thereof with an amount of dicoumarol effective todisrupt performance of the oxidase and production of its reactive oxygenspecies signaling products wherein said dicoumarol is administered incombination with vitamin K.
 6. The method for inhibiting NAD(P)H oxidaseenzymes according to claim 5 wherein the amount of a dicoumarol is from1 mg to 500 mg per day.
 7. The method for inhibiting NAD(P)H oxidaseenzymes according to claim 5 wherein the amount of a dicoumarol is from50 mg to 200 mg per day.
 8. The method for inhibiting NAD(P)H oxidaseenzymes according to claim 5 wherein dicoumarol is administered in anaerosol.
 9. The method for inhibiting NAD(P)H oxidase enzymes accordingto claim 5 wherein dicoumarol is administered orally.